Human cytomegalovirus (HCMV) is an important human pathogen, causing serious disease in immune compromised individuals and in congenitally infected children. Improved methods are needed for prevention and control of HCMV infections. One path for development of novel antiviral agents begins with identification and exploitation of novel intersections between virus and cell biology. An example of this is that HCMV encodes several seven-transmembrane (7TM) proteins that have sequence properties reminiscent of heptaspanning signaling molecules such as G protein-coupled receptors. One of these, pUS17 (encoded by the US17 gene) is expressed in a segmented manner, with its N-terminal domain localizing to the cytoplasm at the periphery of the cytoplasmic virion assembly compartment, and the C-terminal domain localizing to the center of the assembly compartment and to infected cell nuclei. We hypothesize that pUS17 segmentation is the product of regulated intramembrane proteolysis (RIP), and that the N-terminal and C-terminal pUS17 segments have biological roles during infection. RIP is an important mechanism for cellular regulation, for which there are few viral precedents and for which the cellular mechanisms are not fully understood. Thus, this represents a timely opportunity to use a well-evolved viral system to study a cellular mechanism of general importance, and to define the role of this process in infection, so that this information can be used to devise novel antiviral strategies. Thus, we will identify and characterize the mechanisms by which US17 is segmented. This will include (i) determining the boundaries of the segments (Aim 1), and (ii) identifying whether segment biogenesis is due to a proteolytic, translational, or transcriptional process (Aim 2).The significance of the proposed work rests on our identification of a novel mode of intracellular regulation of a virally-encoded 7TM protein that is likely to play an important role in intracellular communication. If there is a proteolysis requirement, this opens a whole new area of antiviral development work. The proposed work will open important new avenues for understanding HCMV biology and its pathogenic mechanisms.